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. 2016 May 7;7(28):43220–43238. doi: 10.18632/oncotarget.9215

Figure 3. Phosphorylation enhances both nuclear localization and activity of Notch1.

Figure 3

A. PC-3 cells were transiently transfected with GFP-tagged wild-type (WT), phosphodeficient (SA) or phosphomimicking (SE) N1ΔE. N1ICD localization was imaged by confocal microscopy also from cells co-transfected with the RFP-tagged Pim1 (P1) and/or treated for 24 h with 0.1% DMSO, 5 μg/ml of DAPT or 10 μM DHPCC-9. B. Shown is the average nuclear localization of N1ICD, as determined from two independent experiments along with the analysed cell numbers (n). C. Equivalent expression levels for the GFP- or RFP-tagged wild-type or mutant proteins were confirmed by Western blotting. D. CSL-dependent luciferase reporter assays were used to detect Notch activity in PC-3 cells transiently overexpressing untagged wild-type or phosphomutant N1ΔE proteins, the equivalent expression levels of which were confirmed by Western blotting. E. Notch wild-type and phosphomutant activities were similarly measured in MCF-7 cells. F. The effects of DHPCC-9 or DAPT treatments on the activity of wild-type Notch1 protein were measured in PC-3 cells. Reporter assays were repeated at least three times and shown are average results from one or more independent experiments.